Characterisation of the effect of stress on nitrogen metabolism in the commercially important agarophyte, Gracilaria gracilis

Doctoral Thesis


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University of Cape Town

Gracilaria gracilis occurs naturally in Saldanha Bay, and was important to the South African export industry as it is a source of two important types of agar, namely food grade and sugar reactive agar. However, a number of abiotic and biotic factors, such as nutrient limitation, has virtually destroyed the G. gracilis resource. An understanding of the physiological response of the alga to nutrient limitation will aid in re-establishing and sustaining G. gracilis populations. However, modelling algal physiology and growth in response to abiotic stresses such as nutrient limitation requires an understanding of the underlying metabolic processes. The present study aimed to address this by investigating nitrogen metabolism and the mechanisms regulating nitrogen metabolism in G. gracilis. This was achieved by profiling changes in gene and protein expression, and activity of two major nitrogen metabolic enzymes, nitrate reductase and glutamine synthetase. Long term culture of G. gracilis in nitrogen replete and lacking conditions indicated that nutrient limitation causes a reduction in intracellular nitrogen and nitrogen protein stores such as phycoerythrin. When various sources were introduced to the culture medium to replenish nitrogen starved G. gracilis, changes in nitrate reductase and glutamine synthetase mRNA, protein and activity seemed to be dependent on the nutrient history of the cells, intracellular and extracellular nitrogen concentrations, metabolites of nitrogen assimilation and other metabolic processes such as carbon metabolism and photosynthesis. Nutrient studies suggested that multiple G. gracilis nitrate reductase and glutamine synthetase isoforms are present and differentially regulated via transcriptional, post-trancriptional, translational and posttranslational mechanisms. Furthermore, the insensitivity of these nitrogen metabolic enzymes to ammonium inhibition and the ability to alter the GS1:GS2 activity ratio possibly represents adaptive strategies developed by G. gracilis to survive nitrogen limitation. Immunocytochemical investigations confirmed the presence of multiple nitrate reductase and glutamine synthetase isoforms. The enzymes were successfully localised to the cell wall, chloroplast and cytosol of G. gracilis. A novel finding was the immuno-localisation of glutamine synthetase to intracellular starch granules. Overall, findings in the current study have suggested multiple roles for these metabolic enzymes that include nitrogen assimilation/transport, cell wall biosynthesis and senescence. This study led to the development of a model of the metabolic changes that occur in nitrogen replete and deplete G. gracilis and provides a firm foundation for future studies of the nitrogen stress response in G. gracilis. Characterisation of the G. gracilis nitrogen stress response may ultimately revive mariculture of this commercially important alga in South Africa.